PI3K／Akt信号通路在骨髓间充质干细胞增殖及成骨分化调控中的作用

目的：研究PI3K/Akt信号通路抑制剂LY294002对骨髓间充质干细胞（mesenchymal stem cells，MSCs）增殖及分化的影响。方法采用贴壁法体外分离人骨髓间充质干细胞（hMSCs），加入PI3K抑制剂LY294002（1、10μmol/L），应用MTT法测定细胞增殖，常规成骨诱导分化培养3或7d，采用碱性磷酸酶（ALP）染色观察成骨分化水平，化学比色法测定ALP活性，茜素红染色后观察矿化钙结节数量并定量分析，Westernblot检测磷酸化Akt蛋白表达，应用Realtime-PCR检测各组细胞BMP2、Runx2、OPN及Osterix等成骨分化标记物的基因表达水平。结果从24至72h，LY294002对hMSCs增殖均产生显著抑制，随时间推延，可见抑制增殖效果增强（P＜0.05）。ALP染色和定量测定提示10μmol/L的ALP活性最强，在不同时间显著高于对照组和1μmol/L组（P＜0.05）。成骨诱导培养3和7d，1、10μmol/L组矿化量都显著高于对照组（P＜0.05）。10μmol/L组矿化量在成骨诱导7d也显著高于1μmol/L组（P＜0.05）。Westernblot检测结果证实成骨诱导可激活Akt磷酸化蛋白表达，但LY294002可抑制该蛋白磷酸化。成骨诱导分化7d，1、10μmol/L均明显促进BMP2、Runx2、OPN、Osterix4种基因mRNA表达（均P＜0.05）。结论PI3K/Akt信号通路参与hMSCs增殖和分化过程。成骨分化伴随下游Akt蛋白表达。PI3K抑制剂可抑制hMSCs增殖，但同时促进其向成骨分化和矿化。

Roles of PI3K/Akt signaling pathway in regulating bone mesenchymal stem cells proliferation and differentiation

Objective To study the effect LY294002, a PI3K/Akt signaling pathway inhibitor, on the prolifer-ation and differentiation of human bone mesenchymal stem cells .Methods Human mesenchymal stem cells （ hMSCs ） were isolated by adherent culture and identified .hMSCs were treated with LY294002 , the PI3K specific inhibitor , at two different concentrations , 1 μmol/L or 10 μmol/L, and the cell proliferation was tested by MTT assay .After 3 days or 7 days osteogenic induction culture , osteogenic differentiation was observed by ALP staining and qualification .Alizarin red staining and determination was adopted to analyze mineralization degree .Western blot analysis was employed for Akt phosphorylation .The gene expressions of osteogenic markers , such as BMP2, Runx2, osteopontin and Osterix , were de-termined by Realtime-PCR.Results In non-induction conditions from 24 hours to 72 hours, LY294002 significantly suppressed hMSCs proliferation and the suppressive effect was increased as time went by （ P〈0.05 ） .10μmol/L showed the highest ALP activity over the control and 1μmol/L groups in ALP staining and quantitation at 3 days or 7 days osteo-genic induction （P〈0.05）.Similarly, 1 μmol/L and 10 μmol/L were more extensively calcified than the control in Alizarin red staining and determination after 3 days or 7 days osteogenic induction （P〈0.05）.At 7 days, 10 μmol/L contained more calcified minerals that 1μmol/L （ P〈0.05 ） .Western blot analysis revealed that osteogenic induction stimulated the phosphorylation of Akt which could be inhibited by LY 294002 . At 7 days osteogenic culture , either 1 μmol/L or 10 μmol/L increased the mRNA expression levels of BMP2, Runx2, osteopontin and Osterix （P〈0.05）. Conclusion PI3K/Akt signaling pathway is involved with the process of proliferation and differentiation in hMSCs .Dur-ing osteogenic differentiation , activation of PI3K/Akt differentially regulates downstream effectors Akt .LY294002 arrests hMSCs proliferation while promots their ost